G-proteins are small regulatory switches that exist in two states: a GTP-bound “on” state, and a GDP-bound “off” state. Methods for determining the activation state of G-proteins are important for understanding G-protein controlled signaling networks.
An important class of G-proteins are ADP-ribosylation factors (ARFs), which regulate cytoskeletal organization, integrin activation or signalling. Cytohesins are known activators of ARFs which catalyze the exchange of ARF-bound GDP for GTP; thus, cytohesin inhibition leads to ARFs remaining in the GDP-bound “off” state.
SecinH3 is a small organic molecule that inhibits the cytohesin class of small (~47 kDa) GEFs. Its application in HepG2 cells suppresses insulin signaling; in mice it causes hepatic insulin resistance.
Here we describe the procedure we used to quantify the amount of activated ARF after stimulation with insulin and incubation with SecinH3, i.e. the amount of ARF loaded with GTP, by pulldown with the N-terminal 316 amino acids of GGA3, a protein that specifically binds to ARF-GTP and not to ARF-GDP.